This is a competitive renewal for CA80964 - Mechanisms that regulate tumor-specific immune responses. The model that we have focused on is an adoptive immunotherapy strategy of B 16BL6-D5 (D5) melanoma. A GM-CSF secreting D5 tumor vaccine (D5-G6) is used to prime T cells in the tumor vaccine-draining lymph nodes (TVDLN) that, following in vitro activation, are adoptively transferred into mice bearing established D5 pulmonary metastases (1). This strategy is closely linked to our clinical efforts of this approach (2, 3) as well as our strong focus on combining GM-CSF and whole cell vaccines for breast, prostate and non-small cell lung cancer (4, 5). In the past 3.5 years we have dissected components of this adoptive immunotherapy model and made considerable progress towards addressing our previous aims. In the course of our studies we discovered an innovative approach to increase priming of tumor-specific CD 4 plus and CD8 plus T cells by vaccinating mice undergoing homeostatic proliferation. Additionally, we made the observation that while CD8 plus T cells alone could eliminate all discernible metastatic disease by day 13, all CD4-deficient animals died of their disease. We see a relationship between these two observations and have formulated aims to address three important questions in the field of tumor immunology that are also directly and immediately relevant to clinical immunotherapy The first question is how do you increase the magnitude of the anti-tumor immune response? This is critical because the failure of immunotherapy is often considered the result of an insufficient immune response. Our answer to this is to create a lymphopenic host, reconstitute that host with lymphocytes and then inoculate with a tumor vaccine. Our recent work with these reconstituted lymphopenic mice (RLM) documents the effectiveness of this strategy that augments priming of both tumor-specific CD8 and CD4 T cells. While we have described (during the past funding cycle) the critical elements for successful vaccination in a normal "intact" host, preliminary studies suggest that the requirements for successful priming in the RLM host are strikingly different. Defining the underlying mechanisms of successful priming in RLM hosts will be the focus of Aim 1. The second question is how does this translate in the setting of established disease? Again preliminary studies suggest that reconstitution with lymphocytes from tumor-bearing mice (TBM) is substantially less effective than that observed in a tumor free model (prelim studies Table 8). Aim 2 will investigate the mechanisms responsible for this effect and develop strategies to overcome it. Question 3 asks what contributions of CD4 divided by T cells are essential to establish a curative immune response following adoptive transfer and whether the CD4 response needs to be tumor-specific? Here we will build on our recent observations (6, 7) and sections C.3 and C.5 of our Preliminary studies to explore this question in Aim 3.